The first year of our NIH-supported studies on the mechanism of intrinsic beta lactam resistance in pneumococci have yielded several important results and in this year's program we would like to continue further studies to follow up each one of these observations: (1) A method for the in vivo labeling and rapid identification of Penicillin Binding Protein (PBP) - in live, growing bacteria - was worked out. With this method we showed that (2) Intrinsic resistance to penicillin in clinically isolated strains of pneumococci from South Africa, Minnesota and Oklahoma have altered PBPs in the direction of greatly decreased affinities for the antibiotic; in contrast, penicillin tolerant pneumococci have normal PBPs. (3) A novel class of tolerant mutants -containing normal level of autolytic activity and yet not lysable by penicillin - have been isolated and designated as LYT plus TOL plus (as contrasted with the more familiar lysis-defective - LYT minus TOL ion -mutants). (4) Intensive search for a plasmid in the multiply drug resistant South African strains yielded negative results. On the other hand, a high-frequency genetic transformation system with heterologous plasmid DNA has been established in pneumococci (5). We also succeeded in finding methods which allow a direct selection for lysis-defect or for chain-formation, i.e., two functions that may be related to penicillin sensitivity (6).